The present invention relates to a sensor using microscopic flexible mechanical structures such as micro-cantilevers, micro-bridges or micro-membranes integrated into microscopic chambers. In particular, the present invention relates to a sensor for measuring biochemical properties of fluids in such chambers.
The measurement of the properties of fluids flowing in microscopic channels is of importance in the field of micro liquid handling systems, which includes systems for measuring:
1) physical properties such as flow rates viscosity and local temperature
2) chemical properties such as pH and chemical composition
3) biological properties such as identification of organic constituents in fluids, including DNA fragments, proteins, and complete biological cells
Microliquid handling systems typically consist of narrow channels of order 100 microns wide and 100 microns deep engraved or embossed into the surface of a thin wafer of a material such as silicon, glass or plastic using reproduction techniques based on micromachining. The surface containing the channels is usually bonded to another surface, in order to seal the channels. Fluids pumped through the resulting channels typically flow in a completely laminar fashion. As a result, several different fluids can be flowed in laminated streams through such microsystems, without any significant mixing of the fluids.
An important advantage of a microliquid handling system is that very small quantities of fluid can be directed in a controlled fashion to various parts of the system, where various analytical techniques can be used to determine the properties of the liquid. This can be done using external analytical techniques such as optical detection. The controlled flow of the fluid is achieved via pumps and valve systems that can be either external or integrated with the microchannels.
Micro-cantilevers are devices where changes in the mechanical properties of a microscopic micro-cantilever are used to detect changes in the environment of the micro-cantilever. The micro-cantilever is typically of the order of 100 microns long, 10 microns wide and one micron thick. The micro-cantilevers are made of a material such as silicon, silicon nitride, glass, metal or combination of any of these, using micromachining techniques. A change in the mechanical properties can for example be a stress formation in the micro-cantilever due to changes in surface stress of the micro-cantilever. Stress formation can also occur due to changes in temperature of the micro-cantilever due to a bimorph effect, if the micro-cantilever is made of two materials with different thermal expansion coefficients. Such stress formations in the micro-cantilever can be detected in a variety of ways. Often the stress formation will result in a deflection of the micro-cantilever. In these situations the deflection can be detected by deflection of a laser light beam by a reflecting surface of the micro-cantilever. Change in the resistivity of a piezoresistor integrated onto the micro-cantilever is another method, which has the advantage that it does not depend on a deflection of the micro-cantilever and it does not require optical access to the micro-antilever.
Change in resonance frequency is another example of a change in a mechanical property. A change in mass of the micro-cantilever can occur if material binds to the micro-cantilever, and such a change will produce a change in the resonance frequency of the micro-cantilever. Such changes can be monitored by actuating the micro-cantilever at a frequency near its resonance frequency, and monitoring changes in the amplitude of the resulting dynamic bending of the micro-cantilever, using methods similar to those described above for the detection of stress formation.
Using these changes in mechanical properties, micro-cantilevers, have been used to detect chemical reactions occurring on the surface of the micro-cantilever, either in gas phase or in liquid phase. For gas phase experiments the measurements have been performed in a gas chamber utilizing optical detection of a micro-cantilever bending. Micro-cantilevers with integrated piezoresistive read-out have been used for thermogravimetry in air. Under ambient conditions the micro-cantilever-based detection technique has proven very sensitive. It has been demonstrated that mass changes down to 0.5 ng and temperature changes down to approximately 10xe2x88x925 C can be resolved. Furthermore, a change of surface stress on the order of 10xe2x88x925 N/m has been detected. In liquids, J. Chen [J. Chen, Ph.D thesis Simon Fraiser University (1995)] reports on a piezoresistive micro-cantilever for mass change detection. Detection of polystyrene spheres was performed in a 3 water tank in which the micro-cantilever was placed. By vibrating the micro-cantilever, changes in the resonance frequency and thereby mass changes of the micro-cantilever could be monitored. The micro-cantilever deflection was monitored by integrated piezoresistive read-out.
PCT patent application WO99/38007 published Jul. 29 1999 describes a system for detecting analytes in a fluid using functionalised micro-cantilevers mounted in a tube. A bending of the micro-cantilever is induced by molecular interactions on one side of the micro-cantilever.The bending is monitored optically by the reflection of a laser beam of the end of the micro-cantilever. Examples of application include the formation of self assembled monolayers (SAM""s) of alkylthiols on a goldcoated micro-cantilever and the partially reversible adsorption of low density lipoproteins. The possibility of testing multiple analytes against multiple analytes is mentioned. A solution for generating a reference signal is proposed exploiting the twisting movement of the micro-cantilever and the ability to distinguish the twisting from the bending movement. Low flow rates are recommended in order to avoid perturbations of the micro-cantilever. This is a clear indication that the envisioned flow system is of macroscopic dimensions.
A micro-cantilever array placed at the top of an open channel has been realised in polymer [C. P. Lee et al., Prooceeding of the xcexcTAS""98 workshop (1998) 245-252; L. P. Lang et al., Sensors and Actuators A 71 (1998) 144-149]. C. P Lee et al. suggest that these micro-cantilevers can be modified for the use of biochemically functionalized tips for use in atomic force microscopy (AFM) or in scanning near field microscopy (SNOM). Hence, this proposed application is related to surface imaging.
Commercially available micro-cantilevers have been used as sensors in liquid. D. R. Baselt et al.[D. R. Baselt et al.,Proceedings of the IEEE. Vol. 85 4 (1997) 672-679] report on piezoresistive micro-cantilevers applied as biosensors using magnetic particles. The coated micro-cantilevers are placed in a liquid cell in which the detection takes place. The micro-cantilevers measure the interaction between particles immobilised on magnetic beads and the immobilised particles on the micro-cantilever surface. If the magnetic beads bind to the surface, the application of a large magnetic field will cause a bending of the micro-cantilever.
U.S. Pat. No. 5,719,324 describes a micro-cantilever based sensor, where a mass change of the micro-cantilever is detected as a change in the resonance frequency of the micro-cantilever. Furthermore, a stress change of a micro-cantilever material is monitored as a micro-cantilever deflection. For mass detection, a piezoelectric actuator oscillates the micro-cantilever and the micro-cantilever deflection is registered by optical read-out. It is mentioned that the mass detection principle can also be applied in liquid.
It is a disadvantage of the above-mentioned systems that micro-cantilever based experiments are carried out in large liquid containers. Such large liquid container systems are very difficult to stabilise thermally. Furthermore, in such large container systems the required volume of chemicals is unnecessary high.
It is a further disadvantage of most of the above-mentioned systems that the micro-cantilever deflection is detected optically. This disadvantage is due to the fact that it may be difficult to obtain optical access to a specific micro-cantileverxe2x80x94especially in the case where a plurality of micro-cantilevers are closely spaced and in the case where the liquid is not transparent.
It is an object of the present invention to integrate micro-cantilevers, micro-bridges or micro-membranes into closed micro-liquid handling systems, in order to provide novel detection mechanisms for monitoring the physical, chemical and biological properties of fluids in such systems.
It is a still further object of the present invention to provide a micro-cantilever, micro-bridge or micro-membrane type sensor having integrated readout. A closed micro-liquid handling system allows laminated flows of different liquids to flow in the channel without mixing, which opens up for new type of experiments and which reduces noise related to the liquid movement.
It is a still further object of the present invention to provide adjacent or very closely spaced micro-cantilevers, micro-bridges or micro-membranes which can be exposed to different chemical environments at the same time by:
Laminating the fluid flow vertically in the micro-channel into two or more streams, so that micro-cantilevers or micro-membranes on opposing sides of the micro-channel are immersed in different fluids, or so that a micro-cantilever, micro-bridge, or micro-membrane is exposed to two different fluids.
Laminating the fluid flow horizontally in the micro-channel, so that micro-cantilevers or micro-bridges recessed to different levels in the micro-channel or micro-membranes placed at the top and at the bottom of the channel are exposed to different fluids.
In this way, changes in viscous drag, surface stress, temperature, or resonance properties of adjacent or closely spaced micro-cantilevers, micro-bridges or micro-membranes induced by their different fluid environments, can be compared.
Neighbouring or very closely spaced micro-cantilevers, micro-bridges or micro-membranes can be coated with different chemical substances using the method just described for immersing adjacent or neighbouring micro-cantilevers, micro-bridges or micro-membranes in different fluids. After coating, the micro-channels can be flushed with other fluids to remove the coating material, and to compare the reactivity of neighbouring or very closely spaced micro-cantilevers, micro-bridges or micro-membranes with different coatings.
It is a still further object of the present invention to provide a micro-cantilever, micro-bridge or micro-membrane based sensor where the liquid volume is minimised in order to reduce the use of chemicals and in order to obtain a system which is easy to stabilise thermally.
The above-mentioned objects are complied with by providing, in a first aspect, a sensor for detecting the presence of a substance in a fluid, said sensor comprising:
means for handling the fluid, said handling means comprising an interaction chamber of micrometer dimensions, an inlet and an outlet,
a first flexible member having a surface, said surface holding a substance, wherein the surface holding the substance is at least partly positioned inside the interaction chamber so that at least part of the substance is exposed to the fluid, and
means for detecting a mechanical parameter associated with the first flexible member, said mechanical parameter being related to the presence of the substance in the fluid.
By micrometer dimension is meant that the interaction chamber has dimensions in the 50-500 microns range (width and depth). The first flexible member may comprise a micro-cantilever having a first and a second end, the first end being attached to the interaction chamber. The micro-cantilever may have a rectangular form and may be approximately 50 xcexcm wide, 200 xcexcm long and 1 xcexcm thick.
The mechanical parameter being associated with the first flexible member may both be a static or dynamic parameter. By static is meant that the flexible member may be subject to a static deformationxe2x80x94e.g. bending. Static deformations are typically induced by stress changes in the flexible member. By dynamic is meant the flexible member may be driven at or near its mechanical resonance frequency. Upon detection of a substance in the fluid the resonance frequency may chance due to a change of mass of the flexible member.
Alternatively, the first flexible member may comprise a micro-bridge having a first and a second end, wherein the first and second ends are attached to the interaction chamber. The dimensions (wide, length and thickness) of a micro-bridge may be similar to the dimensions of the micro-cantilever. Alternatively, the first flexible member may form part of a boundary defining the interaction chamber. The boundary may here be one of the sidewalls of the interaction chamber.
The detecting means for detecting the mechanical parameter associated with the first flexible member may comprise a piezoresistive element, preferably being an integral part of the first flexible member. Preferably, the piezoresistive element forms part of a balanced bridge, such as a Wheatstone bridge. Alternatively, the detecting means may comprise a laser, an optical element and a position sensitive photo detector.
The sensor according to the first aspect of the present invention may further comprise an actuator for moving the flexible member relative to the interaction chamber. The actuator may be implemented in several waysxe2x80x94e.g. by comprising piezoelectric elements, comprising means for providing an electrostatic induced movement, comprising means for providing a magnetic induced movement, or by comprising means for providing a thermal induced movement.
The handling means may be fabricated in a material selected from the group consisting of metals, glasses, polymers or semiconductor materials, such as silicon.
The substance being held by the surface of the first flexible member may be selected from the group consisting of metals, polymers, biochemical molecules or micro-biochemical structures. The group of biochemical molecules and micro-biochemical structures comprises enzymes, DNA, Cells and proteins.
The sensor according to the first aspect of the invention may further comprise a second flexible member being at least partly positioned inside the interaction chamber so that at least part of the second flexible member is exposed to the fluid The sensor may further comprise means for detecting a mechanical parameter associated with the second flexible member. This detecting means may comprise a piezoresistive element being an integral part of the second flexible member. The piezoresistive element may form part of a balanced bridge, such as a Wheatstone bridge.
The second flexible member may serve as a reference to the first flexible member and thereby being adapted to generate a reference signal via the detecting means.
In a second aspect, the present invention relates to a sensor for detecting the presence of a substance in a fluid, said sensor comprising:
means for handling the fluid, said handling means comprising an interaction chamber, an inlet and an outlet,
a first flexible member having a surface, said surface holding a substance, wherein the surface holding the substance is at least partly positioned inside the interaction chamber so that at least part of the substance is exposed to the fluid, and
means for detecting a mechanical parameter associated with the first flexible member, said mechanical parameter being related to the presence of the substance in the fluid, wherein the detecting means form an integral part of the first flexible member.
The first flexible member, the detecting means, the actuator may be implemented as previously mentioned. The interaction chamber may be of micrometer dimensionsxe2x80x94i.e. the 50-500 xcexcm range.
The handling means may be fabricated in a material selected from the group consisting of metals, glasses, polymers or semiconductor materials, such as silicon. The substance being held by the surface of the first flexible member may be selected from the group consisting of metals, polymers, biochemical molecules or micro-biochemical structures. The group of biochemical molecules and micro-biochemical structures comprises enzymes, DNA, Cells and proteins.
In order to obtain a reference signal the sensor according to the second aspect may further comprise
a second flexible member being at least partly positioned inside the interaction chamber so that at least part of the second flexible member is exposed to the fluid, and
means for detecting a mechanical parameter associated with the second flexible member.
Also here the detecting means may comprise a piezoresistive element, said piezoresistive element being an integral part of the second flexible member, and wherein the piezoresistive element forms part of a balanced bridge, such as a Wheatstone bridge.
In a third aspect, the present invention relates to a sensor for detecting the presence of a substance in a fluid, said sensor comprising:
means for handling the fluid, said handling means comprising an interaction chamber, an inlet and an outlet,
a first flexible member having a surface, said surface holding a substance, wherein the surface holding the substance is at least partly positioned inside the interaction chamber so that at least part of the substance is exposed to the fluid, and wherein the first flexible member forms an integral part of the handling means, and
means for detecting a mechanical parameter associated with the first flexible member, said mechanical parameter being related to the presence of the substance in the fluid.
That the flexible member forms an integral part of the handling also means that the flexible member may be fabricated separately and then afterwards being attached to the handling means using a plug-on or snap-on solution. The handling and flexible member may then afterwards be encapsulated to form at least part of the final sensor.
Again, the first flexible member, the detecting means, and the actuator may be implemented as previously described. Also suitable materials for fabrication of the handling means and suitable substances have previously been described.
Furthermore, the detecting means for detecting the mechanical parameter associated with the first flexible member may comprise a laser, an optical element and a position sensitive photo detector.
A reference signal may be generated by a second flexible member being at least partly positioned inside the interaction chamber so that at least part of the second flexible member is exposed to the fluid. The reference signal itself may be generated by a detecting means for detecting a mechanical parameter associated with the second flexible member. The detecting means may comprise a piezoresistive element, said piezoresistive element being an integral part of the second flexible member, and wherein the piezoresistive element forms part of a balanced bridge, such as a Wheatstone bridge.
In a fourth aspect, the present invention relates to a sensor for detecting the presence of a substance in a fluid, said sensor comprising:
means for handling the fluid, said handling means comprising an interaction chamber, an inlet and an outlet,
a first flexible member having a surface, said surface holding a substance, wherein the surface holding the substance is at least partly positioned inside the interaction chamber so that at least part of the substance is exposed to the fluid, and wherein fabrication of the first flexible member is part of fabrication of the handling means,
means for detecting a mechanical parameter associated with the first flexible member, said mechanical parameter being related to the presence of the substance in the fluid.
The fact that the fabrication of the first flexible member is part of the fabrication of the handling means is to be understood in the following way. The fabrication of the handling means involves a plurality of steps. One or more of these step may involve the fabrication of the first flexible member. This issue is addressed in further details in xe2x80x9cDetailed description of the inventionxe2x80x9d.
The first flexible member, the detecting means, and the actuator may be implemented as previously described. Also suitable materials for fabrication of the handling means and suitable substances have previously been described. Also according to this aspect, the sensor may further comprise
a second flexible member being at least partly positioned inside the interaction chamber so that at least part of the second flexible member is exposed to the fluid, and
means for detecting a mechanical parameter associated with the second flexible member.
In a fifth aspect, the present invention relates to a sensor for detecting the presence of a first and a second substance in a fluid, said sensor comprising:
means for handling the fluid, said handling means comprising an interaction chamber of micrometer dimensions, an inlet and an outlet,
a first flexible member having a surface, said surface holding a first substance, wherein the surface holding the first substance is at least partly positioned inside the interaction chamber so that at least part of the first substance is exposed to the fluid,
a second flexible member having a surface, said surface holding a second substance, wherein the surface holding the second substance is at least partly positioned inside the interaction chamber so that at least part of the second substance is exposed to the fluid,
a first detecting means for detecting a first mechanical parameter associated with the first flexible member, said first mechanical parameter being related to the presence of the first substance in the fluid, and
a second detecting means for detecting a second mechanical parameter associated with the second flexible member, said second mechanical parameter being related to the presence of the second substance in the fluid.
The first and second flexible members may comprise a micro-cantilever having a first and a second end, wherein the first end is attached to the interaction chamber. Alternatively, the first and second flexible members may comprise a micro-bridge having a first and a second end, wherein the first and second ends are attached to the interaction chamber. Finally, each of the first and second flexible members may form part of a boundary defining the interaction chamber. This boundary may be a sidewall of the interaction chamber.
The detecting means may comprise piezoresistive elements being integral parts of the first flexible member. The detecting means may also comprise lasers,optical elements and a position sensitive photo detectors.
The sensor may further comprise actuators for the flexible members. These actuators may comprise piezoelectric elements being integral parts of the micro-cantilevers. Other types of actuators may also be applied.
The handling means may be fabricated in a material selected from the group consisting of metals, glasses, polymers or semiconductor materials, such as silicon. The substances being held by the surface of the first and second flexible members may be selected from the group consisting of metals, polymers, biochemical molecules or micro-biochemical structures. The group of biochemical molecules and micro-biochemical structures comprises enzymes, DNA, Cells and proteins.
In a sixth and final aspect, the present invention relates to a sensor for detecting the presence of a first and a second substance in a moving laminated fluid, said laminated fluid comprising, in a cross section perpendicular to a direction of movement, a first and a second region, said sensor comprising:
means for handling the laminated fluid, said handling means comprising an interaction chamber, an inlet and an outlet,
a first flexible member having a surface, said surface holding a first substance, wherein the surface holding the first substance is at least partly positioned inside the interaction chamber so that at least part of the first substance is exposed to the first region of the laminated fluid,
a second flexible member having a surface, said surface holding a second substance, wherein the surface holding the second substance is at least partly positioned inside the interaction chamber so that at least part of the second substance is exposed to the second region of the laminated fluid,
means for detecting a first mechanical parameter associated with the first flexible member, said first mechanical parameter being related to the presence of the first substance in the first region of the fluid, and
means for detecting a second mechanical parameter associated with the second flexible member, said second mechanical parameter being related to the presence of the second substance in the second region of the fluid.
By a moving laminated flow is meant that a measurements may be performed in a continues liquid flow or, alternatively, that the liquid is introduced into the chamber and then temporarily stopped while the measurements are being performed. After the measurements have been performed the liquid is guided away from the chamber.
The detecting means for detecting the mechanical parameters associated with the first and second flexible members may comprise piezoresistive elements being integral parts of the flexible members. Alternatively, the detecting means for detecting the first and second mechanical parameters associated with the first and second flexible member, may comprise lasers, optical elements and a position sensitive photo detectors.
Furthermore actuators may be applied for moving part of the flexible elements relative to the handling means. These actuators may comprise piezoelectric elements, said piezoelectric elements being integral parts of the flexible members. Also with regard to this aspect, the handling means may be fabricated in a material selected from the group consisting of metals, glasses, polymers or semiconductor materials, such as silicon. The substances to by held be the flexible members may be as previously mentioned.
It is an advantage of the present invention that piezoresistors are integrated and used to measure the deflections of the flexible members.
It is a still further advantage of the present invention that a plurality flexible members can be integrated closely together in a micro-system, so that one flexible member can serve as a reference to another, or that nearby flexible members can be immersed in different laminated streams in a fluid flow, so that one fluid can serve as a reference to another.
It is a still further advantage of the present invention that it provides a sensor where the liquid volume is minimised in order to reduce the use of chemicals and in order to obtain a system which is easy to stabilise thermally.
The above object, advantages and features, together with numerous other advantages and features will be evident from the detailed description below of preferred embodiments of the present invention.